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Involvement of Egr-1/RelA synergy in distinguishing T cell activation from tumor necrosis factor-alpha-induced NF-kappa B1 transcription.

Cogswell PC, Mayo MW, Baldwin AS - J. Exp. Med. (1997)

Bottom Line: Characterization of the NF-kappa B1 promoter identified an Egr-1 site which was found to be essential for both the PMA/PHA-mediated induction as well as the synergistic activation observed after the expression of the RelA subunit of NF-kappa B and Egr-1.Furthermore, Egr-1 induction was required for endogenous NF-kappa B1 gene expression, since PMA/PHA-stimulated T cell lines expressing antisense Egr-1 RNA were inhibited in their ability to upregulate NF-kappa B1 transcription.Our studies indicate that transcriptional synergy mediated by activation of both Egr-1 and NF-kappa B may have important ramifications in T cell development by upregulating NF-kappa B1 gene expression.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill 27599-7295, USA.

ABSTRACT
NF-kappa B is an important transcription factor required for T cell proliferation and other immunological functions. The NF-kappa B1 gene encodes a 105-kD protein that is the precursor of the p50 component of NF-kappa B. Previously, we and others have demonstrated that NF-kappa B regulates the NF-kappa B1 gene. In this manuscript we have investigated the molecular mechanisms by which T cell lines stimulated with phorbol 12-myristate 13-acetate (PMA) and phytohemagglutin (PHA) display significantly higher levels of NF-kappa B1 encoding transcripts than cells stimulated with tumor necrosis factor-alpha, despite the fact that both stimuli activate NF-kappa B. Characterization of the NF-kappa B1 promoter identified an Egr-1 site which was found to be essential for both the PMA/PHA-mediated induction as well as the synergistic activation observed after the expression of the RelA subunit of NF-kappa B and Egr-1. Furthermore, Egr-1 induction was required for endogenous NF-kappa B1 gene expression, since PMA/PHA-stimulated T cell lines expressing antisense Egr-1 RNA were inhibited in their ability to upregulate NF-kappa B1 transcription. Our studies indicate that transcriptional synergy mediated by activation of both Egr-1 and NF-kappa B may have important ramifications in T cell development by upregulating NF-kappa B1 gene expression.

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The Egr-1 DNA binding site is required for PMA/PHA–responsive activation and for transcriptional synergy mediated by RelA and Egr-1. (A)  CEM cells were transfected with either the wild-type NF-κB1 promoter (SSLUC) or with each of the site-directed mutants including (a) mNLUC,  which contained a mutated NF-κB site at position −289, (b) mELUC, which contained a disrupted 68-bp Egr-1 site, and (c) mNELUC, which contained mutated NF-κB and Egr-1 sites, and 18 h later cells were stimulated with either PMA/PHA or TNF-α. 24 h after stimulation, cell extracts were  harvested and assayed for luciferase activity. (B) CEM cells were co-transfected with either the SSLUC construct or with mutant reporters (mN, ME,  mNE; 5 μg each) and with various expression vectors (5 μg) including an empty vector control, RelA, Egr-1, or RelA plus Egr-1. Cells were harvested  48 h after transfection and luciferase activities were determined. (C) The SSLUC construct was transfected into CEM cells along with either the vector  control or with an Egr-1 expression construct. 18 h after transfection, cells were either left untreated or stimulated with PMA/PHA or TNF-α. Cells  were harvested 24 h after stimulation and extracts were analyzed for luciferase activity. All transfection assays were performed in triplicate in three independent experiments.
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Figure 3: The Egr-1 DNA binding site is required for PMA/PHA–responsive activation and for transcriptional synergy mediated by RelA and Egr-1. (A) CEM cells were transfected with either the wild-type NF-κB1 promoter (SSLUC) or with each of the site-directed mutants including (a) mNLUC, which contained a mutated NF-κB site at position −289, (b) mELUC, which contained a disrupted 68-bp Egr-1 site, and (c) mNELUC, which contained mutated NF-κB and Egr-1 sites, and 18 h later cells were stimulated with either PMA/PHA or TNF-α. 24 h after stimulation, cell extracts were harvested and assayed for luciferase activity. (B) CEM cells were co-transfected with either the SSLUC construct or with mutant reporters (mN, ME, mNE; 5 μg each) and with various expression vectors (5 μg) including an empty vector control, RelA, Egr-1, or RelA plus Egr-1. Cells were harvested 48 h after transfection and luciferase activities were determined. (C) The SSLUC construct was transfected into CEM cells along with either the vector control or with an Egr-1 expression construct. 18 h after transfection, cells were either left untreated or stimulated with PMA/PHA or TNF-α. Cells were harvested 24 h after stimulation and extracts were analyzed for luciferase activity. All transfection assays were performed in triplicate in three independent experiments.

Mentions: To determine whether Egr-1 is required for PMA/PHA responsive activation of the NF-κB1 promoter, site-directed mutagenesis was performed. Since the NF-κB1 promoter is regulated directly by NF-κB (11, 12), we chose to mutate the −289 κB motif alone or in combination with the −68 Egr-1 binding site. The four luciferase NF-κB1 promoter constructs included (a) SSLUC, the wild-type construct, (b) mNLUC, which contained a mutated NF-κB site at position −289, (c) mELUC, which contained the disrupted 68-bp Egr-1 site, and (d ) mNELUC, which contained mutated NF-κB and Egr-1 sites. As shown in Fig. 3 A, site-directed mutagenesis of the Egr-1 binding element (mELUC) significantly diminished PMA/PHA-responsive activation of the NF-κB1 promoter, but had very little or no effect on TNF-α–mediated stimulation. In contrast, mutating the NF-κB −289 site alone had very little effect on either PMA/PHA– or TNF-α–induced activation (data not shown). These results in conjunction with the promoter deletion studies (shown in Fig. 1 B) would strongly suggest that other κB binding sites (namely −103 and −11) are important for transcriptional activation. Recently, McElhinny et al. (22) demonstrated that the −11 NF-κB site was required for HIV-mediated induction of NF-κB1 promoter in monocytes. Although the −289 motif is probably not the only functional NF-κB motif, site-directed mutagenesis of both the Egr-1 and NF-κB elements (mNELUC), resulted in a further decrease in PMA/PHA-responsive stimulation compared to the mELUC construct (Fig. 3 A). Taking into account that mutagenesis of the Egr-1 site in the NF-κB1 promoter significantly diminished PMA/PHA-responsive stimulation, these results indicate that the −68 Egr-1 site plays an important role in the activation of this gene in T lymphocytes.


Involvement of Egr-1/RelA synergy in distinguishing T cell activation from tumor necrosis factor-alpha-induced NF-kappa B1 transcription.

Cogswell PC, Mayo MW, Baldwin AS - J. Exp. Med. (1997)

The Egr-1 DNA binding site is required for PMA/PHA–responsive activation and for transcriptional synergy mediated by RelA and Egr-1. (A)  CEM cells were transfected with either the wild-type NF-κB1 promoter (SSLUC) or with each of the site-directed mutants including (a) mNLUC,  which contained a mutated NF-κB site at position −289, (b) mELUC, which contained a disrupted 68-bp Egr-1 site, and (c) mNELUC, which contained mutated NF-κB and Egr-1 sites, and 18 h later cells were stimulated with either PMA/PHA or TNF-α. 24 h after stimulation, cell extracts were  harvested and assayed for luciferase activity. (B) CEM cells were co-transfected with either the SSLUC construct or with mutant reporters (mN, ME,  mNE; 5 μg each) and with various expression vectors (5 μg) including an empty vector control, RelA, Egr-1, or RelA plus Egr-1. Cells were harvested  48 h after transfection and luciferase activities were determined. (C) The SSLUC construct was transfected into CEM cells along with either the vector  control or with an Egr-1 expression construct. 18 h after transfection, cells were either left untreated or stimulated with PMA/PHA or TNF-α. Cells  were harvested 24 h after stimulation and extracts were analyzed for luciferase activity. All transfection assays were performed in triplicate in three independent experiments.
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Related In: Results  -  Collection

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Figure 3: The Egr-1 DNA binding site is required for PMA/PHA–responsive activation and for transcriptional synergy mediated by RelA and Egr-1. (A) CEM cells were transfected with either the wild-type NF-κB1 promoter (SSLUC) or with each of the site-directed mutants including (a) mNLUC, which contained a mutated NF-κB site at position −289, (b) mELUC, which contained a disrupted 68-bp Egr-1 site, and (c) mNELUC, which contained mutated NF-κB and Egr-1 sites, and 18 h later cells were stimulated with either PMA/PHA or TNF-α. 24 h after stimulation, cell extracts were harvested and assayed for luciferase activity. (B) CEM cells were co-transfected with either the SSLUC construct or with mutant reporters (mN, ME, mNE; 5 μg each) and with various expression vectors (5 μg) including an empty vector control, RelA, Egr-1, or RelA plus Egr-1. Cells were harvested 48 h after transfection and luciferase activities were determined. (C) The SSLUC construct was transfected into CEM cells along with either the vector control or with an Egr-1 expression construct. 18 h after transfection, cells were either left untreated or stimulated with PMA/PHA or TNF-α. Cells were harvested 24 h after stimulation and extracts were analyzed for luciferase activity. All transfection assays were performed in triplicate in three independent experiments.
Mentions: To determine whether Egr-1 is required for PMA/PHA responsive activation of the NF-κB1 promoter, site-directed mutagenesis was performed. Since the NF-κB1 promoter is regulated directly by NF-κB (11, 12), we chose to mutate the −289 κB motif alone or in combination with the −68 Egr-1 binding site. The four luciferase NF-κB1 promoter constructs included (a) SSLUC, the wild-type construct, (b) mNLUC, which contained a mutated NF-κB site at position −289, (c) mELUC, which contained the disrupted 68-bp Egr-1 site, and (d ) mNELUC, which contained mutated NF-κB and Egr-1 sites. As shown in Fig. 3 A, site-directed mutagenesis of the Egr-1 binding element (mELUC) significantly diminished PMA/PHA-responsive activation of the NF-κB1 promoter, but had very little or no effect on TNF-α–mediated stimulation. In contrast, mutating the NF-κB −289 site alone had very little effect on either PMA/PHA– or TNF-α–induced activation (data not shown). These results in conjunction with the promoter deletion studies (shown in Fig. 1 B) would strongly suggest that other κB binding sites (namely −103 and −11) are important for transcriptional activation. Recently, McElhinny et al. (22) demonstrated that the −11 NF-κB site was required for HIV-mediated induction of NF-κB1 promoter in monocytes. Although the −289 motif is probably not the only functional NF-κB motif, site-directed mutagenesis of both the Egr-1 and NF-κB elements (mNELUC), resulted in a further decrease in PMA/PHA-responsive stimulation compared to the mELUC construct (Fig. 3 A). Taking into account that mutagenesis of the Egr-1 site in the NF-κB1 promoter significantly diminished PMA/PHA-responsive stimulation, these results indicate that the −68 Egr-1 site plays an important role in the activation of this gene in T lymphocytes.

Bottom Line: Characterization of the NF-kappa B1 promoter identified an Egr-1 site which was found to be essential for both the PMA/PHA-mediated induction as well as the synergistic activation observed after the expression of the RelA subunit of NF-kappa B and Egr-1.Furthermore, Egr-1 induction was required for endogenous NF-kappa B1 gene expression, since PMA/PHA-stimulated T cell lines expressing antisense Egr-1 RNA were inhibited in their ability to upregulate NF-kappa B1 transcription.Our studies indicate that transcriptional synergy mediated by activation of both Egr-1 and NF-kappa B may have important ramifications in T cell development by upregulating NF-kappa B1 gene expression.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill 27599-7295, USA.

ABSTRACT
NF-kappa B is an important transcription factor required for T cell proliferation and other immunological functions. The NF-kappa B1 gene encodes a 105-kD protein that is the precursor of the p50 component of NF-kappa B. Previously, we and others have demonstrated that NF-kappa B regulates the NF-kappa B1 gene. In this manuscript we have investigated the molecular mechanisms by which T cell lines stimulated with phorbol 12-myristate 13-acetate (PMA) and phytohemagglutin (PHA) display significantly higher levels of NF-kappa B1 encoding transcripts than cells stimulated with tumor necrosis factor-alpha, despite the fact that both stimuli activate NF-kappa B. Characterization of the NF-kappa B1 promoter identified an Egr-1 site which was found to be essential for both the PMA/PHA-mediated induction as well as the synergistic activation observed after the expression of the RelA subunit of NF-kappa B and Egr-1. Furthermore, Egr-1 induction was required for endogenous NF-kappa B1 gene expression, since PMA/PHA-stimulated T cell lines expressing antisense Egr-1 RNA were inhibited in their ability to upregulate NF-kappa B1 transcription. Our studies indicate that transcriptional synergy mediated by activation of both Egr-1 and NF-kappa B may have important ramifications in T cell development by upregulating NF-kappa B1 gene expression.

Show MeSH
Related in: MedlinePlus